Novel gene therapy agent for haemophilia b and its preparation method

ABSTRACT

The invention deals with the gene drugs for therapy Hemophilia B and its method of preparation. It contains human source gene vector-FIX recombinant constructed using DNA sequence as leading sequence of therapy gene without important physiological function-related gene on the short arms of human group D,G chromosomes or to which DNA sequence is homologous. This invention also provides the method of preparing gene drug. The therapy gene of gene drug for hemophilia B has a high stability of expression, safety, high expression efficiency and no immunogenecity.

SUMMARY OF THE INVENTION

[0001] The invention involves in genetic drug for treatment of hemophilia B and its preparation.

BACKGROUND OF THE TECHNOLOGY

[0002] Hemophilia B also called second type Hemophilia is a kind of haemorrhage, X-linkage recessive hereditary disease due to deficiency of fibrinogen IX (FIX). Its incidence in the male is 1/30000. Human FIX is single-stranded glucoprotein containing 415 amino acid residues, with 56 KD molecular weight. It can be divided into 4 different functional sections from of N-terminal: Gla section (γ-carboxyl glutamic acid section), growth factor section, activating peptide section and catalyze section (or serine protein section). Human FIX is mainly synthesized in liver. Its initial translation product which contains leader sequence with 46 amino acid at the N-terminal turns into mature FIX after modification of deprecursoring peptide, glycosylation and y-carboxylation depending on Vitamin K. FIX exists in blood in the form of zymogen which turns into activated FIX(FIXa) with protease activity by means of activating of FIXa or FVII-tissue factor complex. The content of FIX in plasma in normal people is 5 μg/ml and its half-life of activity is 24 hours (refer to Chuah M K L,DesireCollen &Vanden Driessche. Gene therapy for Hemophilia.J Gene Med 2001;3:3-20).

[0003] FIX is a necessary protein factor in process of endogenetic cruor cascade response. The complex of FIX and regulatory protein accelerates rate of endogenetic cruor cascade response thousands of times, which makes cruor process be finished just in a few minutes. Therefore, when it is short of FIX in human body, it can lead to endless spontaneous or minutely traumatic bleeding, seriously joint distortion and lamness or death due to bleeding in bowel or skull. The human FIX gene was identified in 1982 at chromosome Xq27. 1. hFIX is composed of 8 exons, the coding sequence is 1.383 kb, encoding 415 amino acids. (Choo K H, Goule K G, Rees D J, et al. Nature 1982;299:178-180; Kurachi K, Davie E W. Proc Natl Acad Sci USA 1982;79:6461-6464.). Molecular weight of FIX protein is 56 KD, its concentration in normal plasma is 5 μg/ml(Kaufman R J. Human Gene Therapy 1999; 10:2091-2107).

[0004] Clinical treatment of Hemophilia B is confined to protein substitute treatment also called blood transfusion, or replenishing FIX concentration preparation. However, because FIX's half-life in body is only 24 hours, patients need repeated transfusion or blood preparation to keep life, who will not only undertake heavy economic burden, but also confront infection threat of HIV, HBV and mad cow virus. How to introduce normal FIX gene into patient's body to substitute deficient FIX gene is the key problem of gene therapy for Hemophilia B. At present, vector used for FIX gene therapy research is mainly virus vector, such as retrovirus vector, adenovirus vector and adeno-associated virus vector.

[0005] 1. Kay et al carried out experiment of gene therapy on a dog with Hemophilia B. (refer to Kay M A, Rothenberg S, Landen C N, et al. In vivo gene therapy of hemophilia B: sustained partial correction in factor IX deficient dogs. Science 1993; 262:117-119). The research suggests that RV transferring gene in the liver of big animals possibly have long expression, but the expression is low. It was also found that just 0.1%-1% of liver cells of mouse are transferred by RV in the mice research,(refer to Kay M A, Li Q T, Liu J J, et al. Hepatic Gene Therapy: Persistent expression of human a 1_antitrypsin in mice after direct gene delivery in vivo. Hum Gene Ther 1992; 3:641-647) mainly because RV can't integrate unsplitting cell. So it is necessary to do operation of partial hepatic removal to induce cell of remaining hepatic tissue to split in gene therapy when hepatic cells are used as target cell. Relatively low transferring efficiency in vivo of RV and low retransplanting efficiency of cultured cell in vitro lead to low FIX expression can't thoroughly correct phenotype of Hemophilia B. (refer to Lieber A. Peters M J, Gown A, et al .A modified urokinase plasminogen activator induces liver regeneration without bleeding .Hum Gene Ther 1995; 6:1029-1037; Bowles N E, Eisensmith R C, Mohuiddin R, et al .A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo. Hum Gene Ther 1996; 7:1735-1742; Bosch A, McCray P B, Jr.Chang S M W, et al. Proliferation induced by keratinocyte growth factor e4nhances in vivo retroviral-mediated gene transfer to mouse hepatocytes. J Clin invest 1996; 98:2683-2687). Though retroviral vector can integrate stably into genome of target cell, it only infects splitting cell and has potential danger such as inserting mutagenesis;

[0006] 2. Recombinant Adeno-associated virus (rAAV) vector is more efficient among viral vector of gene therapy of Hemophilia B. It not only makes FIX cDNA express efficiently and stably in receptor, but also is appreciated by many researchers because vector doesn't contain viral gene and is unlikely give rise to cytotoxic T Lymphocyte response.(refer to Jooss K, Yang Y, Fishe K J, et al .Transduction of dendritec cells by KNA viral vectors directs the immune response to transgene products in muscle fibers. J Virol 1998;72:4212-4223).Kay and his colleagues carried out clinical research on mice and a Hemophilia B dog.(refer to Kay M A, Mannno C S, Ragni M V, et al Evidence for gene transfer and expression of factor IX in Hemophilia B patients treated with an AAV vector. Nat Genet 2000;

[0007]24:257-261). It needs further research on human gene therapy because of low titer, complicated preparation and poor largely extension of AVV. In recent years, nonviral vector has been developed and used such as liposome, microcapsules. (refer to Hortelano G, Xu N, Vandenberg A, et al. Persistent delivery of factor IX in mice: gene therapy for Hemophilia using implantable microcapsules. Hum Gene Ther 1999;10(8);1281-1288.

[0008] In recent ten years, many effective measures in gene therapy for Hemophilia B have been taken, but no safe and stable genetic drug with one-off therapy is available. The key problem lies in that no any safety vector without immunogenicity and highly efficient in transferring target cell and long expression of therapy gene in target cell, has been found,.

[0009] The objective of invention is to offer a kind of safe genetic drug for Hemophilia B therapy which can make therapy gene stably express in target cell.

[0010] Another objective of invention is to offer a kind of preparation method of gene drug above.

[0011] Gene drug for Hemophilia B therapy offered by this invention contains vector-FIX recombinant whose leading sequence of objective gene is DNA sequence without important physiological function-related gene on short arm of human group D, G chromosomes or its homologous DNA sequence.

[0012] The applicant found that two families with normal phenotype carrying additional bisatellites microchromosome(BM) which passes down stably among 2 and 3 generations in 2 families, respectively and causes no harm to human body. So the applicant advances a plan of separating BM original and constructing human gene vector. To date, it has been reported such 17 families both at home and abroad, but nobody put forward similar idea using the BM as gene vector. In this study, the applicant confirmed using FISH technique that the BM came from short arms of human group D(13,14,15) and G(21, 22) chromosomes, which are nucleolus tissue section abundant in ribosome DNA (rDNA ) with polymorphism in length (different contents of rDNA ),and gene transcription of the section during interphase of cell division is very active, the applicant infers that the gene in these sites will have high, stable and harmless expression if special DNA fragments isolated from BM can be used as leading sequence of therapy gene and therapy gene can be site-directed into nucleolus tissue section of short arms of human group D,G chromosomes. The following example can give strong proof.

[0013] The inventor at first constructed pUC19 library with BM specific DNA fragments through micro-dissection and micro-cloning technique, and from which single copy fragments which come from the BM and p arms of human groupD, G chromosomes was selected by southern blotting and proved by FISH technique, then acquired a 120 kb specific DNA fragment(BMSF) using single copy fragments as probe to screen human PAC genome and DNA library. The fragments also come from the BM and short arms of human group D, G chromosomes proved by FISH technique (FIG. 1). It doesn't find important psychological function-related gene by analysing BMSF's sequence property. So, it is safe to use it as target site. The invention further used specific DNA fragments of 120 kb or even smaller to construct gene vector.

[0014] The applicant holds that it conforms to objective of invention to use DNA sequence without important psychological function-related gene on short arm of human group D, G chromosomes or its homologous DNA sequence as leading sequence of objective gene. The gene vector constructed is special, which can target therapy gene site-directed into short arms of human group D, G chromosomes and the gene can be highly efficient expressed.

[0015] The prepared drug contains assistant reagents for therapy gene site-directing into host cell, such as liposome and special protein to host cell. According to acquirement of leading sequence of therapy gene, different forms of vectors can be constructed by the existing technique. The methods of constructing vector and introducing therapy gene into vector are common.

[0016] On the base of gaining therapeutic gene leading sequence, different vectors can be constructed .The construction vector method is routine.

[0017] In the case one the invention gives in detail vector-FIX recombinant as showed in SEQ NO.1 in which leading sequence with 3.8 kb comes from BMSF. Positive screening Neo gene is inserted into site 1500 of leading sequence of therapy gene which is divided into two arms with 1.5 kb and 2.3 kb, respectively, using TK as negative screening gene. Inserting position of FIX therapy gene is located in site 5910. The insertion of therapy gene has forward and reversal directions, and the example of invention adopts the latter. FIG. 2 is construction of vector-FIX recombinant.

[0018] The case gives in detail process of constructing vector using specific DNA sequence of 3.8 kb isolated from short arms of human group D, G chromosomes as leading sequence of objective gene. The strain of vector-FIX recombinant acquired asked for preservation to China Typical Culture Conservation Center (China.Wuhan Wuhan University, post code: 430072) on 29^(th) of September, 2000, numbered:

[0019] CCTCC M200003 1. The preservation is named Escherichia coli

[0020] JM109/JH_/pNS_FIX according to assigned clarification of preservation.

[0021] The vector carrying therapy gene can be transferred into cultured human cell, such as fibroblast cell and blood stem cell which also can be used as gene therapy agent for hemophilia B. The example of this invention gives an example of delivering method using existing technique.

[0022] In summary, to correct clinical symptom caused by deficient gene, the cultured human cell with therapy gene transfected or the drug carrying therapy gene vector can be introduced into patient's body by means of hypodermal embed, electroporate or inject intravenously or packaged by liposome respectively, the therapy gene can be stably expressed.

[0023] Effective Experiment of Gene Therapy:

[0024] Experiment in Ex Vivo

[0025] The recombinant human coagulatant FIX-vector was linearized and transfected into HT1080 cell by electroporation, then the positive transfected cell strain was obtained by positive and negative screening. The foreign gene integration was proved by amplifying FIX cDNA flaking intron. Primer pair pNSNeo at Neo gene and 876-7R at outside of targeting arm were used to amplify gDNA from positive transfected cell strain, and a 2.3 kb fragment was obtained, and sequencing analysis indicated the integration is site directed. Two positive cell strains with FIX gene integrated into short arms of human chromosomes 13,21 has fluorescence signal by FISH, which suggests site directed insertion too. Activity result of FIX in positively transfected cell culture reveals that activity of FIX is 3.56 μg/10⁶/24 h and the cell can express stably after passaging in ex vivo for 449 days. The expression product has been proved by Western blotting.

[0026] Experiment in Vivo

[0027] The target vector is human-source vector. There is species diversity between human beings and animals, especially for gene drug. If carrying out target experiment in animal's body, it is uncertain if there is a site-integration as expected and so it is improper to evaluate efficiency and stability of the vector. It is unnecessary to do animal experiment in vivo.

[0028] Toxic Experiment on Gene Drug

[0029] Administration of the gene drug by mice's tail vein, 450 μg/Kg, accounting up to 100 times as high as the single dosage of adult by body weight. The experiment result suggests that the drug is safe by injecting intravenously without toxicity.

[0030] The gene drug for Hemophilia B therapy offered by the invention uses DNA sequence without important psychological function- related gene on p arms of human group D, G chromosomes or its homologous DNA sequence as leading sequence of objective gene to construct gene vector. Undoubtedly, the gene vector constructed can site direct its therapy gene into special target site of host cell, and DNA fragments of the target site don't have important physiological function-related gene, so introduction of therapy gene is safe.

[0031] In one word, because the gene therapy drug for Hemophilia B offered by the invention adopts leading sequence of human-source gene to construct vector with therapy gene, it has below characteristics:

[0032] (1) Stability of expression of therapy gene is good. Because vector with therapy gene can site-insert therapy gene into short arms of human group D, G chromosomes, therapy gene can inherit stably with chromosomes.

[0033] (2) The security is good. The target site of the therapy gene doesn't have important physiological function-related gene, which proves the target site safe. Moreover, vector can site-insert therapy gene into safe target site of cell has been confirmed by FISH (FIG. 3), avoiding random inserting mutation and danger of wild-type virus. So expression of therapy gene at target site is safe. Though the invention doesn't provide clinical cases of gene therapy, cases found by the applicant and researchers both at home and abroad can prove its security from another angle.

[0034] (3) Expressive efficiency is high. Firstly, the vector in the invention contains leading sequence from short arms of human group D,G chromosomes, correspondently, it has 10 target sites in human genome with inserting efficiency 5-10 times high than that of other vectors; secondly, leading sequence is from short arms of human group D,G chromosomes with active gene transcription ,so therapy gene on target position introduced can be expressed high efficiently.

[0035] (4) It has no immunogenicity. The vector is human-source, so it will not generate immunogenicity in human body.

[0036]FIG. 1 is FISH mapping of 120 kb BMSF;

[0037]FIG. 2 is construction of gene vector-FIX recombinant offered by the invention (the whole length of gene vector sequence is 13928 bp); pGEM-7(11033-13928): vector reproductive component and prokaryotic screening system; TK(1-2480): negative screening gene of eukaryotic cell, use TK promoter and TK polyA signal ; Neo (4342-5910): positive screening gene of eukaryotic cell ,use sv40 promoter and sv40 polyA signal; FIX (5911-8677): FIX therapy gene ,use CMV promoter and BGH polyA signal; GLS(2841-4341,8687-11032): leading sequence of therapy gene; Cloning site (5910): inserting position of therapy gene.

[0038]FIG. 3 is FISH mapping of foreign FIX therapy gene in clone of positive cell, which proves the vector can site-direct FIX into short arms of human group D,G chromosomes;

[0039]FIG. 4 is Western blotting result of positive cell, F3-F23 are 6 different positive cell strains, “−” denotes negative control.

[0040] The examples of the invention are only a way of realization of the invention which can't be considered limitation to the invention.

EXAMPLE ONE

[0041] The Preparation Of Gene Leading Sequence

[0042] Obtaining of Clone of Gene Leading Sequence-PAC

[0043] 1.1 BM specific pUC19 library constructed by micro-dissection, PCR and microclone methods ( Deng H X, Yoshiura K, Dirks R W, et al. Hum Genet 1992, 89:13)

[0044] 1.2 Obtaining and identifying of BM specific pUC 19

[0045] (1)Preparation of Bacterial Colony Matrix Membrane

[0046] Draw 14×14 squares on the two nylon membranes marked with A or B signal, and place the two membranes into two dishes contained solid LB medium, respectively, and pick at random 14×12 white colonies from the library dishes and put it into the relevant wells, 100 ng single-copy DNA as positive control in row 13, 100 ng gDNA as negative control in row 14. And then the two dishes are incubated at 37° C. for 10-12 hrs. After taking out the membrane from the dishes, conserve B membrane at 4° C. and deal with A membrane on the filter soaked with solution below and procedure, 10% SDS, 5 min - - - 0.5N NaOH/1.5M NaCl, 3 min - - - 1.5 M NaCl/0.5M Tris.HCl, 3 min - - - 2×SSC/0.2 M Tris.HCl, 10 min - - - vacuum dry at 80° C. for 2 hrs and then conserve it for use at 37° C.

[0047] (2) Preparation of gDNA Probe

[0048] Sample 50-75 ng gDNA, make up ddH2O to 11 ml and boil for denaturing at 100° C. for 10 min, and perform random primer labeling reaction following the system below, 2 m M d NTP (d ATP-), 3 μl, primer mixtture, 2 μl Klenow enzyme, 1 μl α-³² P-d ATP, 3 μl

[0049] vortex the mixture and floating bathe at 37° C. for 30 min. Add 8 μl stop mixture to the system and pass it through G-50 column to pure the probe.

[0050] (3) Hybridization

[0051] The membrane is soaked in 2×SSC solution for 10 min, slightly wipe out the colony pieces on the surface of membrane, and then pre-hybridize in 5 ml hybridization buffer for 30 min. According to the value of liquid scintillation, sample probe solution as the proportion of 1.2×10⁶ cpm/ml hybridization buffer to boil for denaturing at 100° C. for 10 min, and then add 5 ml fresh hybridization buffer to hybridize with the colony matrix membrane at 65° C. for 12 hrs. Wash the membrane as follows: 2×SSC/0.1% SDS, at room temperature for 10 min; 2×SSC/0.1% SDS, 65° C.for10 min; 0.1×SSC/0.1% SDS, 65° C. for 10 min, autoradiograph at −70° C. If the signal of hybridization has none or has slight signals, we can consider it as single copy.

[0052] (4)Sequence and Identification by Southern Blotting;

[0053] Pick the clone on the Membrane B without hybridization signals to amplify at small scale and then extract plasmid DNA for sequencing. Compared to data of Genbank, it is a single copy if they have no similarity. Finally, digest plasmid with EcoRI and separate insert DNA and label with α-³²P-d ATP by random primer method, and hybridize with the gDNA digested with EcoRI enzyme on the nylon membrane as the methods above described, one or two pieces of hybridization band are single copy.

[0054] 1.3 Obtaining and identifying the specific PAC clone of BM and chromosomal short arm of Group D, G.

[0055] (1). Obtaining of positive clone number by scanning human PAC gDNA library label the 260 bp single copy probe P8-7 by Random Primer method with α-³²P-d ATP - - - purification with G-50 column (medium size) - - - conserve at 4° C. for use - - - soak 7 pieces of membranes with 2×SSC buffer for 10 min - - - pre-hybridize at 55° C.for 3 hrs - - - denature the probe at 100° C. for 10 min - - - hybridize with PAC membrane by adding 50 ml hybridization buffer at the proportion of 4.6×10⁵cpm/ml - - - wash membrane: 2×SSC, RT for 10 min once, 2×SSC/0.1% SDS 65° C.10 min twice - - - 70° C. autoradiogragh for 12 hrs - - - develop X-ray film - - - read the positive clone number following the introduction.

[0056] (2) Pick the positive clone number at random on the 5 different plates and buy PAC clone

[0057] 1.4 PAC DNA hybridized with the metaphase cells is confirmed coming from chromosomal short arm of Group D, G by FISH (see FIG. 1).

[0058] The methods above refer to J. Sambrook et al. Molecular Colony. Second Edition. Cold Spring Harbor Laboratory Press. 1989

[0059] 2. Obtaining of Gene Leading Sequence DNA

[0060] Main materials, β-agarase (Bio-Labs), Not, Agarose

[0061] (1) Digested PAC169 with Not I enzyme

[0062] (2) Separate the insert DNA(about 120 kb in size) by PFGE method

[0063] Pulse electrophoresis conditions:

[0064] Electrode buffer: 0.5×TBE, High strength Analytical Grade Agarose (Bio-Rad, Low Melting point Agarose LMP) 1%,

[0065] Switch Time: 2 sec - - - 15 sec.

[0066] Electrophoresis Time: 18 hrs,

[0067] voltage: 6V/cm,

[0068] Angle: 120°,

[0069] Temperature: 14° C.

[0070] (3) Stain with EB solution(0.2 μg/ml) for 30 min after electrophoresis and cut off 3.8 kb (120 kb) gel band according to the marker guiding with sterile knife.

[0071] (4) Deal with the gel with β-agarase and precipitate with alcohol

EXAMPLE TWO

[0072] The preparation of gene drug for hemophilia B which is the recombinant of gene vector-FIX provided by the invention.

[0073] 1. construction of gene vector and transduction of therapy gene

[0074] 1.1 construction of the gene vector

[0075] 1.1.1 Digest PAC DNA with Nsi I and Stu I enzyme(blunt end enzyme), recover 3.8 kb DNA fragment with normal agarose gel and purification with electric elution

[0076] 1.1.2 Digest pGEM-TK vector DNA with Hind III enzyme and fill-in the end to blunt with Klenow enzyme and yield a blunt end.

[0077] 1.1.3 Further digest the product what pGEM-TK/Hind III repair with NsiI enzyme.

[0078] 1.1.4 Link the product purified with 3.8 kb/ Nsi I+Stu I enzyme and the product digested with pGEM-TK/HK+Nsi I at 16° C. for 17 hrs.

[0079] 1.1.5 Thansfer the JM 109 E. coli with the products what linked, and culture in the dish/Amp+ at 37° C.for 18 hrs.

[0080] 1.1.6 Pick the single copy clone at random and double enzyme-cut with pGEM-TK/HK+Nsi I enzyme and identify the positive clone.

[0081] 1.1.7 Ligate Neo/Xba I+Nhe I and pGEM-TK-3.8 KB/Nhe I to construct pNS2 gene vector.

[0082] 1.2 The transduction of FIX gene

[0083] 1.2.1 Clone FIX gene (CDS) into pcDNA 3.1 (−)

[0084] 1.2.2 Design TPCF and TPCR primers and amplify FIX gene and expression component (CMV promoter and BGH poly A signal) to load the two ends with AvrIII enzyme digestion sites; the sequence of primer is

[0085] TpcF: ATgCATCCTAggggAggTCgCTgAgTAgTg

[0086] Avr II

[0087] TpcR; ATgCATCCTAggTACCCCTAgAgCCCAg

[0088] Avr II

[0089] 1.2.3 Digest FIX gene with Avr II enzyme and its expression component (CMV promoter and BGH polyA signal) is ligated with the pNS2 vector digested with Nhe I enzyme.

[0090] 1.2.4 The ligated product was transformed to JM109 E. coli, and the recombinant strain (Collection number is CCTCC M20003 1) was otained.

[0091] The methods above refer to J. Sambrook et al. Molecular Colony. Second Edition. Cold Spring Harbor Laboratory Press. 1989

[0092] 2. Extraction of gene vector DNA

[0093] 2.1 Materials:

[0094] 2.1.1 QIAGE Plasmid Maxi Kit

[0095] 2.1.2 Medium: liquid LB

[0096] Trypton 5 g

[0097] Yeast extract 2.5 g

[0098] NaCl 2.5 g

[0099] ddH2O adjust the volume to 500 ml

[0100] Autoclaved

[0101] 2.1.3 Amp: 100 mg/ml(1000×)

[0102] 2.2 Methods

[0103] 1) Pick positive singe clone into 3 ml LB medium (Amp+)37° C., incubate at 250 rpm for 1 hr

[0104] 2) Add 100 μl primary culture to 100 ml LB medium(Amp+) ,incubate 37° C. at 250 rpm for 16 hrs

[0105] 3) Centrifuge for 15 min at 4° C.to attain pellet and 600 g rotor speed

[0106] 4) Resuspend the pellet with 10 ml Buffer P1

[0107] 5) Add 10 ml Buffer P2 and invert slightly 6 times for 5 min at room temperature

[0108] 6) Add 10 ml pre-cooled Buffer P3, invert slightly 6 times on ice for 20 min

[0109] 7) Centrifuge at 4° C. at 20000 g for 30 min

[0110] 8) Quickly transfer supernatant to a 40 ml of high-speed centrifugation tube and centrifuge at 4° C. at 20000 g for 15 min

[0111] 9) Equilibrate the column QIGEN Tip 500 with 15 ml Buffer QBT

[0112] 10) Filtrate the supernatant through QIGAEtip 500

[0113] 11) Wash the column with 15 ml Buffer QC

[0114] 12) Elute with Buffer QF and collect the elution solution

[0115] 13) Add 10.7 ml(0.7× volume) isopropanol to elution solution and mix up

[0116] 14) Centrifuge at 4° C. ,at 15000 g for 30 min

[0117] 15) Dispose the supernatant and add 5 ml 70% ethanol and centrifuge at 4° C. 15000 g for 10 min to wash DNA precipitation

[0118] 16) Dispose ethanol and air-dry the DNA for 10 min, and then dissolve DNA precipitation with TE buffer

[0119] The recombinant of gene vector-FIX have been preserved in Chinese Type culture Collection Center (Wuhan University, China, postcode: 430072) on 29^(th) of September, 2000,the preservation number is CCTCC M20003 1. The name is Escherichia coli JM1 09/JH-4/PNS-FIX.

EXAMPLE THREE

[0120] In the recombinant of gene vector-FIX in Case Two, FIX gene was transduced into host HTT1080 cells and expressed, the HT1080 cells was preserved in Chinese Type Culture Collection Center (Wuhan University ,China, postcode: 430072) on 18th of August, 2000, The preservation number is CCTCC M20005. The cell strain is named human fibrosarcoma cell strain/JH-1/FIX

[0121] 1. MATERIAL

[0122] 1.1 Cell: HT1080

[0123] Medium; high sugar DMEM+10% FBS (HT1080), EMEM+10% FBS

[0124] 1.2 Electroporation device : Bio-Rad Company

[0125] 2. Methods

[0126] 1) Culture the cells in a 75 cm ² culture bottle and grow up to the 70%-80% confluence

[0127] 2) Harvest the cells and wash twice with HeBs buffer and count the number

[0128] 3) Centrifuge at 4° C. and 15000 rpm for 10 min

[0129] 4) Resuspend the cells with appropriate amount of HeBs buffer to make cells concentration of 10⁶-10⁷ cells/ml

[0130] 5) Take 0.4 ml cuvette and add 0.8 ml cell supernatant and 10 ug DNA vector

[0131] 6) Electric shock at 260V voltage, 550 μF capacitance for 11-13 msec

[0132] 7) Transfer electric shocked cells into 75cm² culture bottle and add 14 ml culture medium with double antibiotics and culture at 37° C., 5% CO₂ for 24-48 hrs

[0133] 8) Add G418 to final concentration of 300 μg/ml to select positive transfected cell, change medium each 2-3 days and add G418 again, using normal cells as control

[0134] 9) Count living clone number in transfected cell when all of normal cells die in seven days, and change the amount of G418 to 150ug/mI to maintain

[0135] 10) Continue to screen transfected cells with final concentration 500 ng/ml GCV

[0136] 11) Assay expression activities of the transferred gene when most of the clones die and add maintain amount of GCV 250 μg/ml to left living cells or non-selected cells are allowed to grow to 70%-80% confluence

[0137] 3.Result

[0138] Transfect the vector to transfer FIX gene to HT1080 cells with electroporation method and attain the positive cell strain after positive and negative screening, the site-directed insertion of gene is confirmed by FISH(see FIG. 3) Activities of FIX gene increase from 0.1 ug/ml to 4.27 μg/ml compared to negative control, what's more, the amount of its expression is still 3.15 μg/ml after 144 days (see Table 1). The products of its expression are confirmed by Western Blotting (see FIG. 4). TABLE 1 Activity detection of FIX gene (μg/10⁶ cells/24 hr) Days after transformed clone F23 35 4.27 72 4.39 100 4.6 111 3.95 139 4.0 144 3.15 449 3.56

[0139] The Certification of Safety

[0140] 1. The case of safety in population of people The applicant has been engaged in research on human and medical genetics since 1973, he found and identified 732 karyotypes which were found first in the world submitted by 470 clinical cytogenetists in 189 labs around China, among these karyotypes there are 41 karyotypes which involve short arms of Group D, G chromosomes. No matter how the fragment translocated to short arms of Group D, G chromosomes originated from which chromosome of chromosome 1-22, or different lengths of fragment from the same chromosome containing one to thousand of genes, the phenotypes of carriers themselves are normal. This finding indicates that the translocation gene to chromosomal short arm in Group D, G can be expressed normally. Therefore, we think it's safe to use short arm in Group D, G chromosomes as target site of gene therapy.

[0141] 1. Karyotype:46,XX,t(1;12;22;15;11;8) (1qter→1p11::8p23→8pter;12pter→12q11::1p11→1pter;22qter→22p11::12q11→12qter;15pter→22p11→22pter; 11pter→11q21::15q15→15qter;8qter→8p23::11q21→11qter)

[0142] phenotype:female, 28 years old, normal phenotype carrier material provider:Wu subing, Cytogenetics laboratory, Department of gynecology and obstetrics, first affiliated hospital of Zhongshan Medical University,Guangzhou

[0143] 2. Karyotype: 46, XY, t(1;13) (1pter→1q32::13p11→13pter; 13qter→13p11:: 1q32→1qter).

[0144] Phenotype:female, 24 years old, normal phenotype carrier Material provider: Xiao Chen, Department of biology, Harbin Medical University,Harbin 150086

[0145] 3.Karyotype: 46, XX, t(2;15) (2pter→cen→15qter ;2qter→cen→15pter)

[0146] phenotype:female,26 years old, normal phenotype carrier material provider:Guo Yuping, et al. Cytogenetics Department, Jiangxi provincial gynecology and obstetrics hospital, Nanchang 330006,Jiangxi province

[0147] 4.Karyotype: 46,XY,t(2;21) (2pter→cen→21pter; 2qter→cen→21qter)

[0148] phenotype:male,32 years old, normal phenotype carrier material provider: Kang Guoqing,et al. Department of genetics, the second affiliated hospital of Shangxi Medical College, Taiyuan 030001

[0149] 5. Karyotype: 46,XY,t(3;21) (2qter→cen→22pter; 3qter→cen→22qter)

[0150] phenotype:male,26 years old,normal phenotype carrier material provider: Gao Yun.Department of toxicology,Bingzhou municipal Medical College, Bingzhou 256603, Shangdong province

[0151] 6. Karyotype: 46,XY,t(3;22) (3pter→cen→22pter; 3qter→cen→22qter)

[0152] phenotype:male,29 years old,normal phenotype carrier material provider: Shi Huajin.Department of genetics,Jingzhou Women and Baby hospital,Jingzhou 121000,Liaoning province

[0153] 7. Karyotype: 46,XX,t(4;15)(4qter→4p13::15p13→15pter; 15qter→15p13::4p13→4pqter )

[0154] phenotype:female,28 years old, normal phenotype carrier material provider: Zhou Ling,et al. Laboratory of genetics,the Wuhan Children hospital, Wuhan 430016,Hupei province

[0155] 8. Karyotype: 46,XY,t(4;21)(4qter→4p15::21p11→21pter;21qter →21p11::4p15→4pqter)

[0156] phenotype:female, 25 years old, normal phenotype carrier material provider: Xu Jinfang, et al. Laboratory of genetics, the sixth people's hospital of Shanghai,Shanghai20000

[0157] 9. Karyotype: 46,XY,t(4;14)(4qter→4q31::14p11→14pter;14qter→14p11::4q31→4qter )

[0158] phenotype: male, normal phenotype carrier material provider: Zhou Mingjun,et al. Xuchang Municipal Central Hospital, Xuchang 161000,Henan province

[0159] 10. Karyotype: 46,XY,t(4;14)(4qter→4q35::14p11→14pter;14qter→14p11::4q35→4qter )

[0160] phenotype: male, 27 years old,normal phenotype carrier material provider: Zhang Xiuquan,et al. Hushan Municipal Women and Nursling Hospital, Hushan 528000,Guangdong province

[0161] 11. Karyotype: 46,XX, t(6;22)(5qter→5q13::22p11→22pter; 22qter→22p11::5q13→5qter)

[0162] phenotype: female, 32 years old, normal phenotype carrier material provider: Zhao Jianping, Anyang Municipal Women and Nursling Hospital, Anyang455000,Henan province

[0163] 12. Karyotype: 46,XY, t(6;22) (6pter→cen6→22qter; 6qter→cen22→22pter)

[0164] phenotype:male,25 years old,normal phenotype carrier material provider: Zhu Xinxia,et al. Laboratory of cytogenetics, Department of Gynecology and Obstetrics, Number 88 Hospital, Taian 271000,Shangdong province

[0165] 13. Karyotype: 46,XY, t(6; 22)(6qter→6p21::22p11.2→22pter; 22qter→22p11.2::6q21→6pter )

[0166] phenotype: male, 33 years old, normal phenotype carrier material provider: Yang Qinglan, Department of Gynecology and Obstetrics, affiliated hospital of Bingzhou Medical College, Bingzhou 256603,Shangdong province

[0167] 14. Karyotype: 45,XX,t(7;21) (7qter→7p22::21p12→21qter)

[0168] phenotype:female,23 years old, normal phenotype carrier material provider: Sun Qingji, et al. Laboratory of genetics, the Wuhan Children hospital, Wuhan 430016, Hubei province

[0169] 15. Karyotype: 46,XY/46XX,t(7;14)(7pter→7q11::14p11→14pter;14qter→14p11::7q11→7qter )

[0170] phenotype: male,28 years old, normal phenotype carrier material provider: Li Luyun, Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0171] 16. Karyotype: 46,XY,t(8;14)(8pter→8p21::14p12→14pter;14qter→14p12::8p13→8pter)

[0172] phenotype: male,27 years old, normal phenotype carrier material provider: Shi Huajin,et al. Department of genetics, Jingzhou Women and Baby hospital,Jingzhou 121000,Liaoning province

[0173] 17. Karyotype: 46,XY,t(9;14)(9pter→cen→14pter; 9qter→cen→14qter)

[0174] phenotype: male,28 years old, normal phenotype carrier material provider: Cheng Qiuyun,et al. Department of reproduction medicine, first affiliated hospital of Hengyang medical college, Hengyang 421001,Hunan province

[0175] 18. Karyotype: 46,XY,t(9;22) (9pter→9p13::22p12→22pter;22qter→22p12::9p13→9pter )mat

[0176] phenotype: female,31 years old, her mother, a young sister of her,a young brother of her and her son have the same phenotype as her, that is normal phenotype carrier material provider: Li Luyun,Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0177] 19. Karyotype:46,XX,t(9;14) (9pter→9q12::14p12→14pter; 14qter→14p12::9q12→9qter).

[0178] Phenotype:female,32 years old, normal phenotype carrier Material provider: Sun Yanyang,et al, Department of biology, Harbin Medical University, Harbin 150086

[0179] 20. Karyotype:46,XX,t(9;15) (9pter→9q21::15p12→15pter; 15qter→15p12::9q21→9qter)mat.

[0180] Phenotype:female,36 years old,normal phenotype carrier material provider: Zhu Guizhen,et al. Laboratory of cytogenetics, Department of Gynecology and Obstetrics, Number 88 Hospital, Taian 271000,Shangdong province

[0181] 21. Karyotype:46,XX,t(10;13) (10pter 10q24::13p11→13pter; 13qter→13p11::10q24→10qter)

[0182] Phenotype:female,28 years old,normal phenotype carrier material provider: Yan Dunqing. Department of Gynecology and Obstetrics,affiliated hospital of Qingdao Medical College,Qingdao266003,Shangdong province

[0183] 22. Karyotype: 46,XX,t(10;13) (10pter→10q24::13p12→13pter; 13qter→13p12::10q24→10qter)

[0184] Phenotype:female,29 years old,normal phenotype carrier material provider: Zhang Yinru,et al. Department of neurology First affiliated hospital of Zhongshan Medical University,Guangzhou510080, Guangdong province

[0185] 23. Karyotype: 46,XX,t(11;14) (11pter→cen→14pter::11qter→cen→14qter)

[0186] material provider:Wang Zhiyong,Department of genetics, Zhacheng County people's hospital,Zhacheng County476200,Henan province

[0187] 24. Karyotype: 46,XX,t(11;21) (11pter→11p11::21p11→21pter; 21qtere→21p11::11p11→11pter)

[0188] Phenotype:female,26years old,normal phenotype carrier material provider: Zheng Jun,et al. Department of genetics,Shanxi provincial women and nursling hospital, Xian710003 Shanxi province

[0189] 25. Karyotype: 46,XX,t(11;15) (11pter→11q13::15p12→15pter; 15qter→15p12::11q13→11qter)

[0190] Phenotype: male,23years old, normal phenotype carrier material provider: Yang Ruifang,et al. Medical center of Obstetrics,affiliated hospital of Shandong Medical University,Jinan250012, Shandong province

[0191] 26. Karyotype :46,XX,t(12;14) (12pter→cen→14pter::12qter→cen→14qter)

[0192] Phenotype:female,28years old, normal phenotype carrier material provider: Han Weitian,et al. Department of eugenics,Liaoning provincial institue of family planing, Shenyang 110031,Liaoning province

[0193] 27. Karyotype:46,XX,t(13;16) (13qter→13p11::16p11.2→16pter;16qter→16p11.2::13p11→13pter

[0194] Phenotype:female,27years old, normal phenotype carrier material provider: An Songlan. Department of genetics, Dalian municipal gynecology and obstetrics, Dalian 110078,Liaoning province

[0195] 28. Karyotype: 46,XY/46,XX,t(13;13) (13qter13p12::13p12→13qter)

[0196] Phenotype: male, 39years old, normal phenotype carrier material provider: Li Luyun, Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0197] 29. Karyotype :46,XY,t(14;18) (14pter→cen→18pter;14qter→cen→18qter)

[0198] Phenotype: male,30years old, normal phenotype carrier material provider: Wang Sugui, et al. Beijing Institute of family planing technology guidance, Beijing 100006

[0199] 30. Karyotype:46,XX,t(14;15) (14pter→14q13::15p13→15pter;15qter→15p13::14q13→14qter)

[0200] Phenotype:female,28years old, normal phenotype carrier material provider: Li Luyun,Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0201] 31. Karyotype:46,XX,t(15qter→cen→22qter)

[0202] Phenotype:female,27years old, normal phenotype carrier material provider: Li Luyun,Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0203] 32. Karyotype:46,XY,t(15; 18) (15pter→cen→18pter;15qter→cen→18qter)

[0204] Phenotype: male,30years old, normal phenotype carrier material provider: Ren Guoqing,et al. Beijing Institute of family planing technology guidance, Beijing 100006

[0205] 33. Karyotype:46,XX,t(15;20) (15pter→cen→2pter; 15qter→cen→2qter)

[0206] Phenotype:female,26 years old, normal phenotype carrier material provider: Wang Xin, et al. Laboratory of genetics, department of obstetrics, the second affiliated hospital, Hunan Medical University,Changsha 410011,Hunan province

[0207] 34. Karyotype:46,XX,t(15;22) (15pter→15q11::22p13→22pter;22qter→22p13::15q11→15qter)

[0208] Phenotype:female,27 years old, normal phenotype carrier material provider: Hu Shengdi, Department of genetics, Hainan provincial people's hospital, Haikou570011,Hainan province

[0209] 35. Karyotype:46,XX,t(15;22) (15pter→15q22::22p11→22pter;22qter→22p11::15q22→qter)

[0210] Phenotype:female,29 years old, normal phenotype carrier material provider:Li Murou, Department of genetics, Xinjiang Medical College, Urumchi 830054

[0211] 36. Karyotype:46,XY,t(16;21) (16pter→16q11::21p11→21pter;21qter→22p11::16q12→16qter)

[0212] Phenotype: male,29 years old, normal phenotype carrier material provider: Zhang Huifang,et al, Institute of family planing technology of Guangdong,Guangzhou510080,Guangdong province

[0213] 37. Karyotype :46,XX,t(18;21) (18pter→cen→21pter; 18qter→cen→21qter)

[0214] Phenotype:female, normal phenotype carrier material provider: Shi Huajin, et al. Laboratory of genetics, Jingzhou women and nursling hospital, Jingzhou 121000,Liaoning province

[0215] 38. Karyotype:46,XX, t(18;21) (18pter→18q11::21p12→21pter;21qter→22p12::18q11→18qter)

[0216] Phenotype: female,26 years old, normal phenotype carrier material provider:Li Xiulin,et al,laboratory of genetics, department of pediatrics,first affiliated hospital of Chinese medical university,Shenyangl 110011,Liaoning province

[0217] 39. Karyotype:45,X,dic(Y;13)(Ypter→Yp1200::13p11→cen→13qter)

[0218] Phenotype: male,4 years old, normal phenotype carrier material provider: Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0219] 40. Karyotype:46,XY,t(Y;15)(15qter→15p12::Yq12→Ypter) pat.

[0220] Phenotype: male,4 years old, normal phenotype carrier material provider: Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0221] Abnormal chromosomes carriers above showed no any abnormal syndrome, which explains that not only nucleolus tissue can receipt foreign genes but also allow foreign genes to express normally.

[0222] 2.Toxicity Test

[0223] Acute toxicity studies of gene drug of the invention

[0224] Objective:

[0225] Investigate the acute toxicity reaction and the conditions of death through vein injection gene drug provided by this invention.

[0226] Materials:

[0227] (1) animals

[0228] Kunming strain mice(n=40);body weight 20.4±1.1 g,the number of both female and male is equal. All of experimental animals and feeds were provided by Experiment animal department of Xiangya Medical School, Central South University. The mice are of eligible certification of medical experiment animals issued by the administrative committee of Hunan provincial medical experiment animals.

[0229] 2 Drugs

[0230] Gene drugs provided in this invention. The sterile water is made up to 11.25 μg/ml injection before experiment.

[0231] Method

[0232] Pretest: 10 little white mice, the number of female and male mice is equal. The gene drug was injected into the mice by the tail vein The dose of gene drug for mice in pretest was 450 μg/kg ( 100 times high as that for adults) ,No toxicity appeared. Formal experiment: According to the characteristics of gene therapy drug, we conducted experiment with pretest dosage. The experimental animals were divided into two groups: test and control groups. The mice were given 450 μg/kg gene drug for test group (equal to 100 times of clinical dose for human being by body weight). The mice in control group were given by the tail vein the equal volume of distillation water (0.4 ml/10 g,a time). Immediately observe the animal reaction to the drug after injected, continuous observation of 14 days, urine and stool situations of animals, common activity and death number were recorded. All of the animal were anatomized and observed the conditions of thorax, abdominal cavity by the end of day 14.

[0233] Results

[0234] The experiment on mice acute toxicity: The control groups mice (n=20) weren't found abnormal activity and death through continuous observing 14 days. The mice appears weary and immovable within 30 minutes after two times injection, and resume normal subsequently. Continuous observing 14 days, the animal uptake, urine and stool, common activity were found no abnormal, and no animal death. There is no significant difference between the test group and control group through pathological examination. The results go as table 2 shows TABLE 2 human gene vector FIX acute toxicity test results concentration vein injection times to clinical general Group n (μg/ml) (ml/10 g/once) dose death number condition control 20 0.4 0 normal FIX 20 11.25 0.4 100 0 normal

[0235] Conclusion

[0236] The vein injection dosage of mice human gene vector FIX (equal to 100 times of clinical dose according to body weight calculation) didn't induce to death .It's indicated no obvious acute toxicity and safe for vein injection administration, suitable for clinical application.

1 3 1 13928 DNA Homo sapiens 1 gggcgaattg ggcccgacgt cgcatgctcc tctagactcg aggaattcta ccgggtaggg 60 gaggcgcttt tcccaaggca gtctggagca tgcgcttaag cagccccgct gggcacttgg 120 cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg 180 gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc 240 ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact 300 agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc 360 ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc 420 gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc 480 ccggcattct gacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc 540 cggcctttcg acctgcagcg acccgcttaa cagcgtcaac agcgtgccgc agatcttggt 600 ggcgtgaaac tcccgcacct cttcggcaag cgccttgtag aagcgcgtat ggcttcgtac 660 ccctgccatc aacacgcgtc tgcgttcgac caggctgcgc gttctcgcgg ccatagcaac 720 cgacgtacgg cgttgcgccc tcgccggcag caagaagcca cggaagtccg cctggagcag 780 aaaatgccca cgctactgcg ggtttatata gacggtcctc acgggatggg gaaaaccacc 840 accacgcaac tgctggtggc cctgggttcg cgcgacgata tcgtctacgt acccgagccc 900 gatgacttac tggcaggtgc tgggggcttc cgagacaatc gcgaacatct acaccacaca 960 acaccgcctc gaccagggtg agatatcggc cggggacgcg gcggtggtaa tgacaagcgc 1020 ccagataaca atgggcatgc cttatgccgt gaccgacgcc gttctggctc ctcatatcgg 1080 gggggaggct gggagctcac atgccccgcc cccggccctc accctcatct tcgaccgcca 1140 tcccatcgcc gccctcctgt gctacccggc cgcgcgatac cttatgggca gcatgacccc 1200 ccaggccgtg ctggcgttcg tggccctcat cccgccgacc ttgcccggca caaacatcgt 1260 gttgggggcc cttccggagg acagacacat cgaccgcctg gccaaacgcc agcgccccgg 1320 cgagcggctt gacctggcta tgctggccgc gattcgccgc gtttacgggc tgcttgccaa 1380 tacggtgcgg tatctgcagg gcggcgggtc gtggcgggag gattggggac agctttcggg 1440 gacggccgtg cccgccccag ggtgccgagc cccagagcaa cgcgggccca cgaccccata 1500 tcggggacac gttatttacc ctgtttcggg cccccgagtt gctggccccc aacggcgacc 1560 tgtacaacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt cccatgcacg 1620 tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg ctgcaactta 1680 cctccgggat ggtccagacc cacgtcacca cccccggctc cataccgacg atctgcgacc 1740 tggcgcgcac gtttgcccgg gagatggggg aggctaactg aaacacggaa ggagacaata 1800 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cgggtgttgg 1860 gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc 1920 gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag 1980 ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaagccctg ccatagccac 2040 gggccccgtg ggttagggac ggggtccccc atggggaatg gtttatggtt cgtgggggtt 2100 attattttgg gcgttgcgtg gggtcaggtc cacgactgga ctgagcagac agacccatgg 2160 tttttggatg gcctgggcat ggaccgcatg tactggcgcg acacgaacac cgggcgtctg 2220 tggctgccaa acacccccga cccccaaaaa ccaccgcgcg gatttctggc gccgccggac 2280 gaactaaacc tgactacggc atctctgccc cttcttcgct ggtacgagga gcgcttttgt 2340 tttgtattgg tcaccacggc cgagtttccg cgggaccccg gccaggacct gcagaaattg 2400 atgatctatt aaacaataaa gatgtccact aaaatggaag tttttcctgt catactttgt 2460 taagaagggt gagaacagag tacctacatt ttgaatggaa ggattggagc tacgggggtg 2520 ggggtggggt gggattagat aaatgcctgc tctttactga aggctcttta ctattgcttt 2580 atgataatgt ttcatagttg gatatcataa tttaaacaag caaaaccaaa ttaagggcca 2640 gctcattcct cccactcatg atctatagat ctatagatct ctcgtgggat cattgttttt 2700 ctcttgattc ccactttgtg gttctaagta ctgtggtttc caaatgtgtc agtttcatag 2760 cctgaagaac gagatcagca gcctctgttc cacatacact tcattctcag tattgttttg 2820 ccaagttcta attccatcag aagctcctta attttatacc actgacttat tttgaaggct 2880 gctataagaa acagccctat gaaactggta ttttcctact gcaaggtggc tactttaaga 2940 caatttttca ttgcattcta tcaagggatg tcttattatt atatcattat atcaagtgat 3000 gttataaata gtaagaatca gattaagggc tcatatgtcc ttctttgtat tgactgttga 3060 aaaggtatgg ggccaaattt gtagtttgtc tggaattaca tatttttggg ggtctctatt 3120 atcttcatac ttatcctatc taaattttcc attgccaaat ttccttactt atttttagtt 3180 ttatcctatt gctcatgtat ttttatgtct ccataagtct attttggaaa aaggcagagt 3240 actcataatt ttagtatatc ttttagcttt atgttgccat aaacctttca ttatatacat 3300 gatcaacaac agcaaattat ctcacttcag tatttagttt attattttac aaactgattt 3360 atgattgcta acatgtaact gaaggtatac actattagaa cacagttttc agtagaaagt 3420 agcactgcca ttgagtaaaa aaatgttcta acattagagc aacattctta tacaagtttg 3480 catgttgttt actgaggtct aaagcatgac tacacaaaag gctgaataaa attcagattc 3540 ttacatacac ataaaattgt tttattgaga tgacaaagta tatttattat gccacccaga 3600 atataatcca ctctgataac tgccagtgta tgcacttgct gaagtaactc agtacataaa 3660 tggtagccac aacagttgct gtgcatgaaa gttcttctct tccagattga agagtgtaca 3720 atctaaagca ttttaaaact ttaaatccct tattagctta aatataattt aaaattttag 3780 tttgccgtac ctataatttg tctgtacact aggttactaa gggtgatatg attacatatg 3840 tggatacaaa ataattttaa tggaaaatga aattagggta ctcaacaaag ataaagggta 3900 atgatcatgt acactaaccg tatttgagat tagtttaagc ctggggtagc tatacttatg 3960 tttcacagac cttgagaaga tagggaaaaa aagcttttat caacattgct aaggaacagg 4020 taaaagctaa cattaggtaa ctaagaggtg acataaaaaa gactgaataa aatatcatgg 4080 aggtttcata ataagattgg aaattccata gactaggaga gaaaagatcc caaaatatac 4140 atgctcattg ggaaaacagc tagtaagaac aaggagagat ctctatttaa tgatacaata 4200 gtagagttat aatttcctgt atattgtaaa tttcaagcat ttaaacattt tcattgaatt 4260 ataaaatatt atttgtaaaa gaaagaaaaa cagcacaact gcagattaca gatgactaag 4320 atagatgaat catgaaaagg tgctagattg tgagcggata acaatttcac acaggaaaca 4380 gctatgacca tgattacgcc aagctctcga cgggatcgcg gccgcgatcc agacatgata 4440 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt 4500 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt 4560 ggggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc 4620 ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg 4680 tgatggcagg ttgggcgtcg cttggtcggt catttcgaac cccagagtcc cgctcagaag 4740 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa 4800 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc 4860 aacactatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa 4920 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga 4980 tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc 5040 tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct 5100 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc 5160 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac 5220 aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca 5280 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc 5340 tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc 5400 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag 5460 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt 5520 tcaaccatgg tggatcgatc caagctccca acacaactat gtcagaagca aatgtgagga 5580 gcaactgatc ctacctcacc ttatatgctc tgccctggct cctgccctct ctatcctgtg 5640 tgagcagatt ggcccttacc aaggtgtggc tctacggaat caggcttcgg tgatgacaag 5700 catatttctc cctagaatgc tgtgccactc actggcttag gagtctcagc tctgggtact 5760 ccctctgaat aatgtttgtc cttatctgtg cagagaacac tgtctctaaa gcatcctttt 5820 tggcaacgca tttgctcaat caactactga attggtgtta aaattaattt tccttttttt 5880 ctcattatgc aaataagaaa ttgagaagca aagctagggg aggtcgctga gtagtgcgcg 5940 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt 6000 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga 6060 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg 6120 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc 6180 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat 6240 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat 6300 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 6360 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 6420 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac 6480 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa 6540 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt 6600 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact 6660 gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgt 6720 ttaaacgggc cctctagact cgagcggccg ccactgtgct ggatatctgc agaattccac 6780 cacactggac tagtggatcc gaccttacca ctttcacaat ctgctagcaa aggttatgca 6840 gcgcgtgaac atgatcatgg cagaatcacc aggcctcatc accatctgcc ttttaggata 6900 tctactcagt gctgaatgta cagtttttct tgatcatgaa aacgccaaca aaattctgaa 6960 tcggccaaag aggtataatt caggtaaatt ggaagagttt gttcaaggga accttgagag 7020 agaatgtatg gaagaaaagt gtagttttga agaagcacga gaagtttttg aaaacactga 7080 aagaacaact gaattttgga agcagtatgt tgatggagat cagtgtgagt ccaatccatg 7140 tttaaatggc ggcagttgca aggatgacat taattcctat gaatgttggt gtccctttgg 7200 atttgaagga aagaactgtg aattagatgt aacatgtaac attaagaatg gcagatgcga 7260 gcagttttgt aaaaatagtg ctgataacaa ggtggtttgc tcctgtactg agggatatcg 7320 acttgcagaa aaccagaagt cctgtgaacc agcagtgcca tttccatgtg gaagagtttc 7380 tgtttcacaa acttctaagc tcacccgtgc tgaggctgtt tttcctgatg tggactatgt 7440 aaattctact gaagctgaaa ccattttgga taacatcact caaagcaccc aatcatttaa 7500 tgacttcact cgggttgttg gtggagaaga tgccaaacca ggtcaattcc cttggcaggt 7560 tgttttgaat ggtaaagttg atgcattctg tggaggctct atcgttaatg aaaaatggat 7620 tgtaactgct gcccactgtg ttgaaactgg tgttaaaatt acagttgtcg caggtgaaca 7680 taatattgag gagacagaac atacagagca aaagcgaaat gtgattcgaa ttattcctca 7740 ccacaactac aatgcagcta ttaataagta caaccatgac attgcccttc tggaactgga 7800 cgaaccctta gtgctaaaca gctacgttac acctatttgc attgctgaca aggaatacac 7860 gaacatcttc ctcaaatttg gatctggcta tgtaagtggc tggggaagag tcttccacaa 7920 agggagatca gctttagttc ttcagtacct tagagttcca cttgttgacc gagccacatg 7980 tcttcgatct acaaagttca ccatctataa caacatgttc tgtgctggct tccatgaagg 8040 aggtagagat tcatgtcaag gagatagtgg gggaccccat gttactgaag tggaagggac 8100 cagtttctta actggaatta ttagctgggg tgaagagtgt gcaatgaaag gcaaatatgg 8160 aatatatacc aaggtatccc ggtatgtcaa ctggattaag gaaaaaacaa agctcactta 8220 atgaaagatg gatttccaag gttaattcat tggaattgaa aattaacagg gcctctcact 8280 aactaatcac tttcccatct tttgttagat ttgaatatat acattctatg atcattgctt 8340 tttctcttta caggggagaa tttcatattt tacctgagct gaagcttaag tttaaaccgc 8400 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 8460 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 8520 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 8580 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct 8640 tctgaggcgg aaagaaccag ctggggctct agggggtacc tagcagagat ttctatcaca 8700 cctatcaggg atacacaatt tccaagaatt tcagaagtgt ttggtgttcc tattaacata 8760 aatccggaaa taacacctga gtgaactgtc ttctaattct tcaactggat ggctttttag 8820 tgtaaaagat gttgaatact gattgacttt ttaataattt tatagtatat gtcagaaata 8880 ttgcacagtc cctatttaca tcattctaca gtggttttta aaatgtttta agaataaaaa 8940 acatgaaaac tttatttgat ttttctgagg aaataacttt ttggatttaa tttcaatgaa 9000 accgttgata acatttccct ccccaacaat ctctggcaac gatccctcag attttaatga 9060 ttatgtatta ttacctttta atacaagtag aataacactc agggaattta caacatttgt 9120 tattttcagt aaatacattg gttgaagttt aaaagtctat ccgtagtaaa cttacatctt 9180 tcaggagctt ggtcaatgtg ttctggacaa agcaggaaga tgtgactgaa atcctgaaag 9240 gagccggctc ctgcagcaca aggataatga tacatctggg tacatttctc ttcacagcat 9300 ttgatagtgg ctccaaagtg cttacaaaat gcacattgct gaaaggggta aaggagagaa 9360 atctctttat aaaaccttga aaaggaatat ttaaatataa gctgggaagg tataaaaaac 9420 tctctgtaac atcacaagta aacaaattga acctgcaaaa tattaaacaa aggattcatt 9480 aaaaataata aaatctacat tactcaattt agtgctttgt gtgctaccaa ctcatccttc 9540 cattcaaatt agaaagttag aatttcattc cttatatttt caaaaataaa ttgtgaagca 9600 ttttagaaac aaaacctaaa attttttttt aaaagcaaat agtaatatgg ttaaaggggc 9660 aggtttctat attgaggatt attataaagt ttttaaatcc taccaaaact agtaatagga 9720 acatatatta tttatgagac atattactat tttttaccct gcctaaaaat aaatacaaat 9780 aaattcatca attataagtt aacagggaca caaatggtta aagactcaca cacaaaaaaa 9840 acaaaactac atacttcaat gtagcaatca acttcaaatt tcttaacaaa agatggaaat 9900 gttggggaaa aaattagtca tctggtatct ttcccatttc aacctgcctc cattatcttg 9960 caagtggtaa aatgcacaga aataagcctc aaacaagagg ggcagtctag ggcaagtgaa 10020 cacataagtc ggaagaaatt atgtaaaatg ttgcatttac ttattcagtt ttcccttaga 10080 atgattcaca aactcttcct cattctccca agtccatttt gagtatcatt ttctttgaag 10140 agagtctgat gggccctgta ctatacagta tgaaatctct ctgtgggaaa tgactatcta 10200 acataaattt ttgtttacac cgttacatgg tacctacttg cttatgccat tacatgatca 10260 gtttaccttt ttctcaacct aatccaagat ccttcaattg aggcactata ctatctttgt 10320 atccaaagca ccaaaaatgc tgcttcaaac aggccctaat agataggtgt tcctatacat 10380 ataccaaaaa gacttaactt ttggtgatct tgtttgtgag tgtggctcat aaacagctta 10440 gttgagataa ctggagcctc atgtagcaga gacagttgga ccctgctaac attactgtgg 10500 atatcttcac atgttactac attgacttta tattctgcta attaaccagg gactacagta 10560 gttaaaatta taattgtttt caatgtttta tgtgtaaatc tgtatctcac atactatcaa 10620 actcttcctc actgtcatca gtctactgca ttgaatccaa cataacaaag ctaaatgact 10680 cctgagggct gaatcagaaa gaagaaaaga aagagataca aaactttagt cggcccggtg 10740 gctcacacct gtaatcccag cactttggaa ggccaaggcg ggcggatcac gaggtcagga 10800 gatcgagacc atcctggctg atacagtgaa actccatctc tactgaaaat acaaaaaatt 10860 agctggacgt ggtggtgggc acctgtagtc ccagctactc aggaggctga agcaggagaa 10920 gcttctaaat aactcataaa cactaattac tgttgtgaca ctttaatttt atacaatatt 10980 tataagtata cagaataaca tttcagtgct attttggcac tcaagggtat taatgcatag 11040 cttgagtatt ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc 11100 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 11160 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 11220 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 11280 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 11340 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 11400 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 11460 ccgtaaaaag gccgcgttgc tggcgttttt cgataggctc cgcccccctg acgagcatca 11520 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 11580 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 11640 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 11700 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 11760 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 11820 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 11880 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 11940 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 12000 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 12060 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 12120 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 12180 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 12240 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 12300 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 12360 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 12420 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 12480 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 12540 gcgcaacgtt gttggcattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 12600 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 12660 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 12720 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 12780 cttttctgtg actggtgagt actcaaccaa gtcattctga gaataccgcg cccggcgacc 12840 gagttgctct tgcccggcgt caatacggga taatagtgta tgacatagca gaactttaaa 12900 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 12960 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 13020 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 13080 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 13140 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 13200 aggggttccg cgcacatttc cccgaaaagt gccacctgta tgcggtgtga aataccgcac 13260 agatgcgtaa ggagaaaata ccgcatcagg cgacgcgccc tgtagcggcg cattaagcgc 13320 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 13380 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 13440 aaatcggggg ctccctttag ggttccgatt tagagcttta cggcacctcg accgcaaaaa 13500 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 13560 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 13620 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 13680 gttaaaaaat gagctgattt aacaaatatt taacgcgaat tttaacaaaa tattaacgtt 13740 tacaatttcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc 13800 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta 13860 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac 13920 tcactata 13928 2 30 DNA Artificial Sequence Primer sequence TpcF 2 atgcatccta ggggaggtcg ctgagtagtg 30 3 28 DNA Artificial Sequence Primer sequence TpcR 3 atgcatccta ggtaccccta gagcccag 28 

1. Gene drug for Hemophilia B contains gene vector-FIX recombinant constructed by DNA sequence as leading sequence of therapy gene without important psychological function related gene on the short arms of human group D, G chromosomes or to which DNA sequence is homologous.
 2. Gene drug for Hemophilia B according to the claim 1 wherein the said vector has the DNA sequences as SEQ NO. 1, and the insert site of FIX therapy gene is
 5910. 3. Gene drug for Hemophilia B according to the claim 1 or 2 which contains in vitro human cells which contain the vector-FIX recombinant as the Right claim I and II claim.
 4. Preparation method of gene drug for therapy hemophilia B contains following procedures: (1) construct the gene vector using the DNA sequences without important physiological function-related gene or to which DNA sequence is homologous on the short arms of human group D,G chromosomes as the leading sequence for gene therapy. (2) Transduce the FIX therapy gene into the above gene vector to obtain the vector-FIX recombinant.
 5. Preparation method of gene drug for therapy hemophilia B according to claim 4 wherein: (1) The leading sequence of gene therapy select from DNA sequence without importance physiological function- related gene DNA Sequence, length of 3.8 Kb ,insert positive selection gene Neo into site 1500 of leading sequence of therapy gene, yielding two arms of 1.5 kb and 2.3 kb using TK as negative selection gene on the short arms of human D,G groups chromosomes construct gene vector. (2) The inserting site of the FIX gene therapy is
 5910. 6. Preparation method of gene drug for therapy hemophilia B according to claim 4 or 5 wherein transferring the vector-FIX recombinant into in vitro human cell. 